Platelets are defined by their essential roles in hemostasis and the formation of pathological thrombi. Therefore, the major focus of platelet research is focused on identifying and developing safer and more effective anti- thrombotic therapies. A second goal is to have the ability to predict the effectiveness of specific therapies across populations. Pharmacogenomics may allow us to predict who will respond to or be resistant to therapies. In order for this to be successful, there needs to be a clear relationship between specific polymorphisms and physiological output. Finally, the alleles need to be present at high enough frequency to warrant testing prior to the start of therapy. In the current project w will examine common polymorphisms of a platelet receptor (PAR4) to explain the differences in reactivities and response to an antagonist at the molecular level. We will determine the structural rearrangement of PAR4 upon activation by thrombin using structural mass spectrometry (amide hydrogen deuterium exchange (HDX), histidine HDX, and hydroxyl radical foot-printing) with purified PAR4 and PAR4 on cells and platelets. These studies will determine how the tethered ligand influences the overall conformation of the receptor. Pharmacological studies in human platelet show that the PAR4 sequence variants have dramatically different responses. For example, PAR4-120T is hyper-reactive and PAR4-296V is resistant to signaling. We will determine how the PAR4 variants affect the ligand binding site and the transition to the active conformation at the molecular level. Further, PAR4-296V suppresses the activity of other alleles suggesting that it forms dominant negative homodimers. We will examine the physical interaction between PAR4 variants, PAR1 and P2Y12 to determine how these receptors influence signaling pathways. Finally, we will determine how the PAR4 sequence variants influence platelet reactivity in acute coronary syndrome patients on the current standard of care (aspirin and a P2Y12 antagonist) and if the altered platelet reactivity due to PAR4 variants is exacerbated with the PAR1 antagonist vorapaxar. By analyzing the structural rearrangements of PAR4 following activation by thrombin, these studies will provide the first detailed description of the tethered ligand activation mechanism for the PAR family and have the potential to uncover allosteric sites that can be exploited therapeutically. More specifically, understanding how naturally occurring sequence variants influence PAR4's response may allow us to predict the most appropriate antiplatelet therapy for patients depending on their genotype.